Numerous genes have been identified in invertebrate organisms such as Drosophila melanogaster, Caenorhabditis elegans and Sacharomyces cerevisiae which participate in developmental events. In mammalian systems, only a few genes which play an integral role during development have been identified (Blau, (1988) Cell 53:673). For example, the muscle-specific genes, MyoD1 and myogenin, have served as important models of genetic control of murine differentiation. Somites and limb buds first express myogenin and MyoD1 on days 8 and 10 of gestation, respectively (Sasson, et al. (1989) Nature 341: 303-308) and are known to control the switch to the myoblast lineage in vitro (Davis, et al. (1987) Cell 51: 987-1000). The Hox-5 gene complex exhibits an interesting pattern of expression in murine limb buds after the 9th day of gestation (Dolle, et al. (1989) Nature 342: 161-111), although the function(s) of the encoded genes have yet to be defined. Few other genetic markers are known which tag cells in early and intermediate stages of murine development. For example, no gene has been associated uniquely with the initial process of segmentation, which occurs on day 8 of gestation in the mouse (for review, see Rossant and Joyner, (1989) Trends in Genetics 5: 277-283).
Immortalized and fully transformed cells frequently transcribe genes which are expressed in (and presumably influence) normal mammalian development (reviewed, Ruddon, (1987) Gene Derepression in Cancer Cells, in Cancer Biology, Oxford University Press, New York, pp 431-436). In some cases, these "oncofetal" genes do not appear to contribute to the neoplastic phenotype. For example, a-fetoprotein is expressed by trophoblast cells and by many tumor cells. In other cases, developmentally regulated genes play a primary role in the conversion of cells to the transformed phenotype. For example, the proto-oncogenes c-myc, c-src, c-fos, and c-fms are expressed during embryonic development, and have been shown to regulate developmental steps in vitro (for review, see Adamson, (1987) Placenta 8: 449-466).
In the development of the immune system, T lymphocytes are derived from precursor stem cells which enter the thymus to undergo differentiation and maturation. Many genes are either activated or repressed as the T cell passes through different stages of development within the thymus. For example, the cells acquire the IL-2 receptor, CD4, and/or CD8 on their surface during this time. These differentiation markers are important for T cell development and/or function. Many gene products are increased in their levels of expression in developing T lymphocytes. These include the T cell receptor for antigen as well as the markers CD4 and CD8. Many other antigens have served as T cell markers before their exact function in lymphocytes were known. Only recently has it been discovered that the T cell antigen Pgpl aids thymocytes in their homing to the thymus whereas T200 (CD45) serves as a component for intracellular signalling. Another T cell marker, Thyl, still has no known function associated with it.
The SL 12.4 cells exhibit a CD4/CD8 double negative phenotype and therefore resemble thymocytes at a relatively early stage of development. Furthermore, they do not express the T cell receptor alpha subunit. SL 12.4 cells, however, can be induced to stably express CD4 and CD8 on their surface after co-cultivation upon thymic epithelial monolayers. TCR-alpha mRNA is also induced after these treatments. Thus, it appears that SL 12.4 cells have the capacity to undergo differentiation and maturation. This unique in vitro biological system mimics, to some extent, the thymic microenvironment.
A number of genes have been identified which are first expressed in developing thymocytes. Many of these genes encode proteins which must be expressed for T cell precursors to become functional in the immune system, for example: 1 ) the TCR for antigen which is required for antigen recognition; 2) CD25 (the IL2 receptor) which must be expressed for the cells to respond to the cytokine IL2; 3) gene products important for signal transduction during antigen recognition, such as CD3, CD4, CDS, CD45, 4) some of the gene products involved in thymocyte homing to target organs, and 5) gene products involved in T cell activation (Fowlkes and Pardoll, Advances in Immunology 44:207-264 (1989); Hood et al., (1985) Cell 40, 225-229; Rothenberg and Lugo, Develop. Biol. 112, 1-17 (1985); Adkins et al., Ann. Rev. Immunol. 5:325-365 (1987); Crabtree, Science 243:343-355 (1989); Kwon and Weissman, Proc. Natl. Acad. Sci. USA 86:1963- 1967 (1989). There is remarkable heterogeneity in thymocyte subsets which express different combinations of expressed genes. Gene expression has been analyzed in detail in many, but not all, of the numerous classes of thymocytes and it is likely that genes remain to be identified that encode products which function in T cell development and homing; particularly those which are expressed in numerically infrequent, transient progenitor thymocytes.
Due to the extensive heterogeneity of thymocytes, it is not feasible to obtain fractionated progenitor thymocytes in sufficient numbers or purity to fully characterize the cascade of gene expression which occurs during development. For this reason, lymphoma and leukemia cell lines have been used extensively to study gene expression in lymphoid development (Greaves, (1986) Science 234:697-704; Hanley-Hyde and Lynch (1986) Ann. Rev. Immunol. 4:621-649. A considerable body of literature indicates that numerically infrequent, transient progenitor cells are the target of transformation to malignancy; and further that some of the characteristics of the transformed target cells are preserved in the tumor cells. Unexpected gene expression in tumor cells was frequently dismissed as an aberration of transformation. However, careful analysis of "aberrant" gene expression in hematopoeitic tumor cells, has revealed rare subsets of normal progenitor cells which express such genes (Greaves, (1986) Science 234:697-704; Hanley-Hyde and Lynch (1986) Ann. Rev. Immunol. 4:621-649; Pierce and Speers (1988) Cancer Res. 48:1996-2004).
The heterogeneity of murine and human lymphoma cell lines derived from a single individual can result from differences in the extent of maturation reached by individual cells. The heterogeneity of established T lymphoma cell lines has been utilized to obtain closely related cell clones which differ in a limited number of characteristics. Hedrick, et al (Hedrick, et al., (1984) Nature 308:149-153), using subtraction cloning techniques, provided estimates that T and B, cells differ in the expression of about 100 genes. It is likely that closely related T lymphoma cells might differ in the expression of even fewer genes. Such cell clones provide an opportunity to work with pure populations of cells with defined and stable phenotypes which differ in a limited number of characteristics. The SL12 T lymphoma model system was developed and utilized in the present application to provide such a closely related cell population. (Hays et al., (1986) Int. J. Cancer 30:597-601; MacLeod, et al., (1984) Cancer Research 44:1784-1790; MacLeod, et al., (1985) J. Nat. Cancer Inst 74:875-882; MacLeod, et al., (1986) Proc. Natl. Acad. Sci. USA 83:6989-6993; Siegal, et al., (1987) J. Exp. Med. 166:1702-1715).
SL12.4 cells are similar to thymocytes at an intermediate stage of maturation (Fowlkes and Pardoll, (1989) Advances in Immunol. 44: 207-264). The two cell clones differ in their biological properties. SL12.4 cells generate extranodal tumors and are sensitive to glucocorticoid-induced lysis, whilst SL12.3 cells cause diffuse disseminated tumors resistant to lysis by glucocorticoids.